Subdiffraction Fluorescence imaging of Bone Biomechanics

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DESCRIPTION (provided by applicant): The hypothesis that mechanically induced distortions of components of murine cortical bone tissue can be measured with 5 nm resolution or better using subdiffraction quantum dot imaging will be tested. The ultimate aim is to study the dynamics of loading of bone tissue with a particular focus on deformation within osteocyte structures and pericellular matrix components. The results will be coupled with finite element analysis models to estimate strain distributions. In the initial experiments murine cortical bone mineral and matrix collagen will be stained chemically or immunochemically using quantum dot labels with different emission wavelengths. The concentrations of the labeling reagents will be adjusted to guarantee that the tissue is sparsely (about 1 label every 2-5 microns) labeled, so that fluorescence results from single dots. Nanometer motion will be observed by fluorescence microscopy as changes in center of gravity of the diffraction spot of a dot, using a high NA objective and an ultrasensitive electron-multiplied CCD imager. Mineral and matrix relative motions will be correlated under normal loading and in the plastic deformation region. The emission wavelengths of the labels will be chosen to optimize the trade-offs between minimization of bone tissue autofluorescence, camera quantum efficiency and emission spectrum band width. To colocalize quantum dots of different emission wavelengths they will be attached to photocleavable reagents, brought to the same point on the tissue and then separated with a brief pulse of UV light. Our candidate photocleavable linker is bis(p-aminophenyl) disulfide, which will allow colocalization of two different quantum dots. Alternatively, labels will be applied randomly and the correlations obtained by statistical analysis of the matrix of observed movements. With success of this phase of the project, extension to labeling cytoskeletal elements will be undertaken.